Molecular Characterization of a Catalase-Negative Staphylococcus aureus Blood Culture Isolate

Abstract

Here we report a catalase-negative methicillin-sensitive Staphylococcus aureus isolate collected from a blood culture. Sequencing through the gene encoding catalase, katA, demonstrated a 2-bp insertion. The resulting frameshift mutation generates a protein that has lost 26 amino acids (aa) at its C-terminal domain.

CASE REPORT

An 86-year-old nondiabetic male with chronic renal failure had a prosthetic arteriovenous (AV) polytetrafluoroethylene (PTFE) graft inserted in the right thigh for dialysis. The operation was complicated by superficial wound infection and immediate graft thrombosis, which was managed conservatively. The graft was left in situ. Subsequently, he presented with purulent discharge from the operative site and was started on vancomycin empirically. He underwent immediate graft removal, and frank pus was noted along the whole length of the graft intraoperatively. Postoperatively, the patient recovered uneventfully.

The local ethics committee deemed that ethics review was not required for this case report (National Healthcare group Domain Specific Review Board application number 2015/00590).

Aerobic cultures of the infected graft grew no bacteria. Blood cultures taken at the same time as the graft removal gave positive results. The positive blood culture was plated on Trypticase soy agar with 5% sheep blood. After 24 h of incubation at 35°C, smooth and creamy β-hemolytic colonies were seen. Gram staining of the culture preparations showed clusters of Gram-positive cocci characteristic of staphylococci. The routine procedure on the blood culture bench entails performing two supplementary phenotypic tests (tube coagulase and catalase production) in addition to matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) to ensure the most accurate identification. The isolate gave a coagulase-positive test result suggestive of S. aureus but was repeatedly negative in the catalase slide test performed with 3% H₂O₂. MALDI-TOF MS identified the isolate as S. aureus with a high level of confidence. Antibiotic susceptibility was determined using the Etest (bioMérieux, Marcy l'Etoile, France), and breakpoints were defined according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The isolate was susceptible to oxacillin, cefoxitin, linezolid, daptomycin, vancomycin, clarithromycin, erythromycin, gentamicin, doxycycline, and rifampin. The molecular identification of S. aureus was confirmed with rpoB sequencing (1), with the isolated strain having 99% identity to S. aureus MSHR1132 (GenBank accession number FR821777.2). A species-specific PCR also confirmed that the isolate was S. aureus (2).

Full-length catalase gene (katA) sequencing was performed using the primer set comprising 5′-ATGTCACAACATGATAAAAA-3′ and 5′-TTATTTTTTAAAGTTTTCGTA-3′. The primers were designed using S. aureus MSHR1132 (GenBank accession number FR821777.2) catalase as a reference. This yielded an amplicon of 1,518 bp. Sequencing analysis revealed the presence of 7 silent mutations, A75T, A78G, T306A, T477C, G708A, A732T, and A951T, and a 2-bp insertion (CA) after nucleotide position 1157 compared to the katA of S. aureus MSHR1132. The insertion is predicted to create a frameshift resulting in the production of a truncated protein of 479 aa instead of the full-length enzyme of 505 aa.

Multilocus sequence typing (MLST) was performed using modified primers for aroE (3), glpF, gmk, tpi, and yqiL (4). Allele and sequence type (ST) assignments were made by comparisons to the S. aureus MLST database (http://saureus.mlst.net/). The isolate was of ST2250, belonging to clonal complex 75 (CC75). Genome sequencing of staphylococci of this lineage has shown them to be phylogenetically divergent from typical S. aureus strains (5). Initial descriptions of CC75 isolates came from analyses of S. aureus skin and soft tissue infections in indigenous communities in the Northern Territory of Australia (6). spa sequence typing was performed using the Ridom StaphType spa sequencing protocol (http://www.ridom.de/staphtype/spa_sequencing.shtml). The isolate was assigned a spa type of t5078 at the Ridom SpaServer (http://www.spaserver.ridom.de/). Staphylococcal cassette chromosome mec (SCCmec) typing was carried out as previously described (7). No SCCmec elements were detected, consistent with its methicillin sensitivity.

The susceptibility of S. aureus to H₂O₂ was determined (8). Methicillin-sensitive NCTC (National Collection of Type Cultures) S. aureus 10788 was used as a catalase-positive control. Increased sensitivity to H₂O₂ was observed in the catalase-negative isolate, with kill curves indicating that the isolate had an approximately 60% survival rate compared to the catalase-positive counterpart (data not shown).

For most clinical microbiology laboratories, MALDI-TOF MS is the dominant method of bacterial identification. However, as no diagnostic test is perfectly accurate, phenotypic tests are still relied upon to complement and corroborate identifications made by MALDI-TOF MS. As such, catalase production remains an important supplementary biochemical test for the differentiation of staphylococci from streptococci (catalase negative). All Staphylococcus species produce catalase except for S. aureus subsp. anaerobius and S. saccharolyticus (9). Catalase-negative S. aureus strains have been sporadically reported and represent an atypical minority of isolates implicated in human infection. The earliest observation of catalase-negative S. aureus was in 1976 (10), but it was only in the last decade, through molecular studies, that deficiencies in catalase production were correlated to mutations in katA. Table 1 summarizes the katA mutations detected in various catalase-negative isolates. The mutations identified are seemingly random, with no particular bias toward a particular mutation type.

TABLE 1.

Key characteristics of catalase-negative Staphylococcus aureus specimens reported between years 2007 and 2015

Report no.	Specimen	Type of mutation	Effect of the mutation on catalase	Reference or source
1	Tracheal secretion	Frameshift	Truncated protein of 462 aa	11
2	Sputum	Frameshift	Truncated protein of 225 aa	12
3	Abscess	Frameshift	Truncated protein of 21 aa	13
4	Excised mitral valve	Nonsense	Truncated protein of 267 aa	14
5	Ulcer	Missense	Loss of active site residing at His58	15
6	Blood	Frameshift	Truncated protein of 479 aa	This study

A typical catalase is formed by four identical monomeric subunits, each containing in its active center a heme group and NADPH (16). Each monomer is composed of four distinct structural regions, including the N-terminal arm (approximately residues 1 to 55), an antiparallel β-barrel forming the core of the subunit (approximately residues 55 to 301), a wrapping domain (approximately residues 302 to 416) that links the β-barrel, and the α-helical domain of the C terminus (approximately residues 417 to 484) (16). Functional analysis of Escherichia coli catalase via site-directed mutagenesis demonstrates that a complete C-terminal domain is indispensable for efficient folding into an active and stable enzyme (17). We hypothesize that the requirement of the C terminus for activity can also be extended to the S. aureus catalase. In our case, the truncation of the protein to 479 aa eliminates the C terminus and hence is likely the cause of loss of catalase activity.

Catalase is critical for oxidative stress resistance. It protects the bacterium from oxidative damage by reactive oxygen species (ROS) by catalyzing the decomposition of hydrogen peroxide (H₂O₂) to water and oxygen. Consequently, catalase is postulated to be a virulence factor in bacterial pathogens that operates by conferring protection from ROS generated by host phagocytes. There is clear evidence that staphylococcal catalase protects the bacteria from H₂O₂-mediated killing of macrophages, thereby contributing to intracellular persistence (18). Catalase has also been demonstrated to be essential for the intracellular survival of bacteria such as Mycobacterium tuberculosis (19), Helicobacter pylori (20), and Leptospira interrogans (21).

The role of S. aureus catalase in virulence is less clear. Some researchers have observed a correlation between virulence and catalase activity (8, 22, 23), and yet others have not found any evidence of such a correlation (24, 25). Nevertheless, clinical isolates of catalase-negative S. aureus appear to have a role in human infections (11–15, 26). Clonal outbreaks of catalase-negative S. aureus infections have been reported, suggesting that such strains are as virulent as wild-type strains (27, 28).